One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior

Abstract

For more than 100years, cells and tissues have been studied in vitro using glass and plastic surfaces. Over the last 10-20years, a great body of research has shown that cells are acutely sensitive to their local environment (extracellular matrix, ECM) which contains both chemical and physical cues that influence cell behavior. These observations suggest that modern cell culture systems, using tissue culture polystyrene (TCP) surfaces, may fail to reproduce authentic cell behavior in vitro, resulting in "artificial outcomes." In the current study, we use bone marrow (BM)- and adipose (AD)-derived stromal cells to prepare BM-ECM and AD-ECM, which are decellularized after synthesis by the cells, to mimic the cellular niche for each of these tissues. Each ECM was characterized for its ability to affect BM- and AD-mesenchymal stem cell (MSC) proliferation, as well as proliferation of three cancer cell lines (HeLa, MCF-7, and MDA-MB-231), modulate cell spreading, and direct differentiation relative to standard TCP surfaces. We found that both ECMs promoted the proliferation of MSCs, but that this effect was enhanced when the tissue-origin of the cells matched that of the ECM (i.e. BM-ECM promoted the proliferation of BM-MSCs over AD-MSCs, and vice versa). Moreover, BM- and AD-ECM were shown to preferentially direct MSC differentiation towards either osteogenic or adipogenic lineage, respectively, suggesting that the effects of the ECM were tissue-specific. Further, each ECM influenced cell morphology (i.e. circularity), irrespective of the origin of the MSCs, lending more support to the idea that effects were tissue specific. Interestingly, unlike MSCs, these ECMs did not promote the proliferation of the cancer cells. In an effort to further understand how these three culture substrates influence cell behavior, we evaluated the chemical (protein composition) and physical properties (architecture and mechanical) of the two ECMs. While many structural proteins (e.g. collagen and fibronectin) were found at equivalent levels in both BM- and AD-ECM, the architecture (i.e. fiber orientation; surface roughness) and physical properties (storage modulus, surface energy) of each were unique. These results, demonstrating differences in cell behavior when cultured on the three different substrates (BM- and AD-ECM and TCP) with differences in chemical and physical properties, provide evidence that the two ECMs may recapitulate specific elements of the native stem cell niche for bone marrow and adipose tissues. More broadly, it could be argued that ECMs, elaborated by cells ex vivo, serve as an ideal starting point for developing tissue-specific culture environments. In contrast to TCP, which relies on the "one size fits all" paradigm, native tissue-specific ECM may be a more rational model to approach engineering 3D tissue-specific culture systems to replicate the in vivo niche. We suggest that this approach will provide more meaningful information for basic research studies of cell behavior as well as cell-based therapeutics.

Keywords: Cell culture; Cell microenvironment; Differentiation; Extracellular matrix; Mesenchymal stem cells (MSCs); Niche.

Fig. 1

Proliferation of MSCs and cancer cells on the three different culture substrates. Cells were grown for 4 days on the three culture substrates (TCP, BM-ECM, and AD-ECM), released from the surfaces, and then counted after trypan blue staining. The data are presented as the number of cells per cm². (A) BM-MSCs or AD-MSCs were cultured on the three substrates. (B) Cancer cell lines were cultured on the three substrates. *P < 0.05, vs. TCP; ***‡P < 0.05, vs. BM-ECM.

Fig. 2

Culture substrate affected MSC spreading morphology. BM- and AD-MSCs were cultured on TCP, BM-ECM, and AD-ECM for 24 h and spreading morphology measured. (A) Quantification of cell circularity on the various substrates. *P < 0.05, vs. TCP; ‡P < 0.05, vs. BM-ECM. (B) Morphology of the MSCs on the three substrates using brightfield microscopy.

Fig. 3

Osteogenic (OB) and adipogenic (AD) differentiation of BM-MSCs was directed by culture surface. (A) OB differentiation of the BM-MSCs was visualized using Alizarin Red staining for calcification and AD differentiation was visualized using Oil Red O staining for lipid droplets. (B) Quantification of pixel staining, as a measure of differentiation efficiency, was performed on 15 randomly-selected areas for each substrate. *P < 0.05, vs. TCP; ‡P < 0.05, vs. BM-ECM.

Fig. 4

Protein composition of BM- and AD-ECM determined by mass spectrometric analysis. (A) The principle collagen components of both ECMs were types VI, XII and I. (B) Protein components of BM- and AD-ECM that are shared in common or unique.

Fig. 5

Principle architectural differences between BM- and AD-ECMs as revealed by microscopic imaging. (A) Second-harmonic generation microscopy, (B) collagen type VI immunofluorescent staining, and (C) atomic force microscopy.

Fig. 6

Fiber orientation differences between BM- and AD-ECMs. Data were fit to a normal distribution, with 90^° corresponding to the mode of the observed orientations. The fit-line of the distribution is overlaid on the raw data. (A) Fibrous structures in BM-ECM displayed densely-organized and highly-oriented fibers (σ = 11.3), while AD-ECM fibers in (B) exhibited a broader range of orientations (σ = 35.6). Mean fiber orientation measurements were performed on 15 randomly-selected areas of BM- and AD-ECM (70 μM × 70 μM) atomic force microscopy images.

Fig. 7

Surface topography of BM- and AD-ECMs. Measurement of surface roughness (R_a) was performed using atomic force microscopy on 15 randomly-selected areas measuring 70 μM × 70 μM. The roughness of TCP was too low to be measured using this method. * P < 0.05, vs. TCP; ‡P < 0.05, vs. BM-ECM.

Fig. 8

Mechanical properties of BM- and AD-ECM. Storage modulus was measured by performing four mechanical tests on each ECM using small angle oscillatory shear rheology (SAOS). ‡P < 0.05, vs. BM-ECM.

Fig. 9

Aqueous contact angle and surface free energy of TCP, BM-ECM and AD-ECM. (A) Water (ddH₂O), glycerol and toluene formed unique contact angles on the three substrates. Data represent 8 measurements for each solvent on each substrate. (B) Oss–Chaudhury–Good analysis revealed considerable differences in surface energy between the three substrates. * P < 0.05, vs. TCP; ‡P < 0.05, vs. BM-ECM.